Acquisition of high-resolution images from within internal organs using endoscopic optical imaging has several clinical applications. In particular, endoscopic optical coherence tomography (OCT) capable of visualizing tissue microstructures is emerging as a promising tool for detection, diagnosis, and monitoring of disease in luminal organs. However, difficulties associated with optical aberrations and the trade-off between transverse resolution and depth-of-focus significantly limits the scope of applications. This work presents a new class of optical imaging catheters termed nano-optic endoscopes that address the difficulties associated with current endoscopic imaging catheters. We incorporate a metalens with the ability to modify the phase of incident light at sub-wavelength level into the design of an OCT catheter to achieve near diffraction-limited imaging through negating non-chromatic aberrations. A metalens consists of a 2-dimentional array of subwavelength-spaced scatterers with specific geometric parameters and distribution that locally shift the phase of the incident light and modify its wavefront. The metalens ability to arbitrarily and accurately modify the phase allows the nano-optic endoscope to overcome spherical aberrations and astigmatism, the essential barriers to diffraction-limited imaging. Remarkably, the tailored chromatic dispersion of the metalens in the context of spectral interferometry is utilized to maintain high-resolution imaging beyond the input field Rayleigh range, overcoming the compromise between transverse resolution and depth-of-focus. Endoscopic imaging is demonstrated in resected human and swine airway specimens and in sheep airways in vivo. The versatility and design flexibility of the nano-optic endoscope significantly elevate endoscopic imaging capabilities that will likely impact clinical applications.

Optical imaging techniques generally offer shallow penetration depths due to high scattering in biological tissue. We have recently developed dual-axis optical coherence tomography (DA-OCT) for interferometric imaging at extended depths. By illuminating and collecting at oblique angles, multiply forward scattered photons from deeper penetration depths were favorably detected. A Monte Carlo study demonstrated a >12-fold increase in signal-to-background ratio (SBR) in skin tissue at 1 mm depth by DA-OCT. A 1300 nm DA-OCT system was designed and constructed, offering an imaging depth of up to 2 mm in skin tissue

A 1060 nm optically pumped tunable VCSEL was formed from an InGaAs/AlGaAs/GaAs half-VCSEL bonded to a MEMS movable mirror on a silicon substrate. The VCSEL was co-packaged in a 14-pin butterfly module with an 825 nm pump laser and a 1060 nm semiconductor optical amplifier. The co-packaged device exhibited shot-noise-limited sensitivity with up to 50 mW output power and 75 nm tunability. Ophthalmic OCT, especially whole-eye imaging and ocular biometry, is considered the primary application of this device. However, we have also investigated LiDAR to greater than 10 meter ranges with non-mechanical beam steering through angular diffraction from a grating. A new generation of photonic integrated circuit LiDARs work this way and we have investigated the depth resolution limitations due to time dispersion from the grating. Distributed fiber temperature sensing was also demonstrated.

Optical coherence tomography (OCT) is a noninvasive imaging modality which can provide cross-sectional imaging of the tissues in high-resolution. Especially in retina imaging, the OCT becomes one of the most valuable imaging tools for the diagnostics of the eye diseases. Considering the scattering and absorption properties of the eye, the 1000 nm OCT system is preferred for the retina image. In this study, we describe an akinetic swept source OCT system based on pulse-modulated active mode locking (AML) fiber laser at the 1080 nm wavelength region for in-vivo human retina imaging. The akinetic AML wavelength swept fiber laser is constructed with polarization maintaining fiber which has average linewidth of 0.625 nm, a spectral bandwidth of 81.15 nm and a duty ratio of 90 % without buffering method. We successfully obtained in-vivo human retina images using proposed OCT system without the additional k-clock and the frequency shifter providing wide field of view of 43.1º. The main retina layers such as RPE can be distinguished through the OCT image with axial resolution of 6.3 m.

Biological functions rely on local microvasculature for delivering oxygen and nutrients and carrying away metabolic waste. Alterations to local oxygenation level are manifested in diseases including cancer, diabetes mellitus, etc. The ability to in vivo quantify oxygen saturation (sO2) of single vessels down to capillary level to assess local tissue oxygenation and metabolic function is highly sought after. Visible light optical coherence tomography (vis-OCT) has shown promise in reaching this goal. However, to achieve reliable measurement in small vessels are challenging due to the reduced signal and requires data averaging to improve the spectral data quality. Therefore, a method to quality control the vis-OCT data from small vessels becomes essential to reject unreliable readings. In this work, we present a generalized method with several quantitative metrics to evaluate the spectral data for reliable sO2 measurements. Parameters of the scanning protocol and the statistical data cleaning can be flexibly adjusted according to different applications and system performances. We used this method to measure sO2 of C57BL/6J mice lower extremity microvasculature and validated it via introducing hyperoxia for expected sO2 changes. After validation, we applied this method on C57BL/J mouse ear microvasculature to conduct in vivo single capillary OCT oximetry. This work intends to standardize the data processing method for in vivo oximetry in small vessels by vis-OCT.

We propose a novel method to visualize the integrated birefringence information for polarization-sensitive optical coherence tomography (PS-OCT) of biological tissue. A strategy that integrates the comprehensive birefringence property in a resultant image is introduced to obtain high contrast images of the birefringent samples. Then, color-based automatic segmentation of birefringent components from 3D scanned tissue volume is proposed to isolate the 3D network of the nerve bundles in a whole mouse brain. Experimental validation and demonstrations are given by imaging ex vivo mouse tail and whole brain tissues to show the usefulness of proposed comprehensive birefringent imaging and segmentation methods. These results sufficiently demonstrate the practical usefulness of the proposed strategy of using comprehensive polarization as the imaging parameter in the PS-OCT imaging of biological samples, indicating potential applications in both pre-clinical and clinical environments where accurate identification of birefringent tissue components is important, for example the nerve identification in delicate surgical remove of the diseased tissue mass in surgery.

In Optical Coherence tomography (OCT), dispersion mismatches cause degradation of the image resolution and are, thus, compensated accordingly. However, dispersion is specific to the material that is causing the effect and can, therefore, carry useful information regarding the composition of the samples. In this summary, we propose a novel technique for estimating tissue dispersion by calculating the cross-correlation of images acquired at different center wavelengths to estimate the shift between their features, also known as walk-off, and use that to calculate the dispersion. Since a distinct reflector is not required, this method is applicable to any sample and can even be implemented in vivo and in situ in human tissues. The proposed technique was verified ex vivo resulting in Group Velocity Dispersion (GVD) values comparable to those obtained from estimating the walk-off from a mirror, as described in the literature. The applicability to cancer diagnosis was evaluated on a small set of gastrointestinal normal and cancer OCT images. Using the statistics of the GVD estimates, tissue classification resulted in 100% sensitivity and 81% specificity (92% correct classification rate). The success of these preliminary results indicates the potential of the proposed method, which should be further investigated to elucidate its advantages and limitations.

We applied quantitative dynamic full-field OCT (qDFFOCT) to imaging of human induced pluripotent stem cell retinal organoids which are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histological analysis and immunofluorescence staining in which multiple specimens fixed at different times are used to reconstruct developmental processes, qDFFOCT imaging can provide repeated images and analysis of the same living organoids with a contrast created by intracellular organelle motion and linked to metabolism. In order to quantify the dynamic signal, we computed each image in Hue-Saturation-Value color-space and benefitted from the latest advances in GPU computing to accelerate the process. We performed time-lapse acquisitions in a locked plane, highlighting cell differentiation, division and mitosis with a sub-micrometer resolution. By moving deeper into the samples, we were also able to acquire series of planes in depth to reconstruct the organoid 3D organization. We also applied qDFFOCT on a damaged macaque cornea and used cutting edge algorithms to track cell motion and successfully reconstruct a migration map of epithelial wound healing. This could help understand the healing mechanism and have great interest in cell therapy. Besides showing our latest results we will explain the signal processing chain we developed to compute quantitative dynamic images where the colors code continuously for dynamic frequencies. Our overall aim is to use the dynamic signal as a non-invasive marker to predict cell type and cell cycle phases, making qDFFOCT a new label-free imaging method.

A Gabor-domain optical coherence microscope (GDOCM) with 2-micrometer invariant lateral and axial resolutions and a working distance of 15 mm was developed for 3D imaging of corneal tissue over a 1 mm2 field of view. The increased working distance over the previous in-contact implementation enables imaging of corneal tissue inside the viewing chamber in which corneas are stored after recovery from donors. The GDOCM system was used to image excised human corneas. 3D images of the cornea were acquired by imaging through the PMMA viewing chamber. The images achieved cellular resolution in the volume being imaged. Due to the curvature of the cornea, the endothelium, a single layer of cells lining the posterior surface of the cornea, cannot be viewed in a single en face image. A flattening algorithm was implemented to obtain an en face view of the endothelium. The GDOCM images were compared with those acquired with a specular microscope commonly used in eye banks for endothelial evaluation, and the endothelial cell density was assessed for both sets of images. A key advantage of GDOCM is the capability to image the entire thickness of the cornea in 3D with cellular resolution over a large field of view.

Over the past 5 years, visible light Optical Coherence Tomography (OCT) has emerged as a promising technique for ultrahigh resolution microstructural imaging and depth-resolved imaging of chromophores. In the retina, visible light OCT can simultaneously induce and observe retinal changes during the phototransduction cascade, including bleaching-related absorption changes, as well as intrinsic scattering, cell swelling, and possible longer-term changes in retinal chromophores. Here we investigate outer retinal reflectance changes during visible light OCT in mice to better understand the contributions of these various signals.
All experiments were performed on pigmented (C57BL/6J) and albino (BALB/c) mice in an initially dark-adapted state. There were no consistent reflectance changes in any layers including and proximal to the External Limiting Membrane (ELM). However, reflectance increased in the inner segment / outer segment (IS / OS) junction and outer segments tips (OST) of both strains. Layers distal to the photoreceptors such as the Retinal Pigment Epithelium (RPE), Bruch’s membrane (BM), and choroid showed a consistent increase in pigmented mice and showed no significant change in albino mice. Though our results are qualitatively well-explained by results from photopigment bleaching and intrinsic optical signal experiments in the literature, the time scale of some of the changes observed in our study is too long, which could indicate either signals with a different physiological origin or the need for a more precise model to describe imaging and stimulation using the same beam profile.

In this study, we demonstrate a novel scanning pattern for improving flow quantification in optical coherence tomography angiography (OCTA) with a high scanning efficiency. A bidirectional interleaved scan pattern was introduced to adjust the adjacent inter-scan time in order to achieve OCTA sensitivity to different flow speeds. This bidirectional scanning protocol uses a triangular function on the fast scanning direction, meaning that it takes the same time in completing B-scans at adjacent lateral positions, acquired in opposite directions. By applying this scheme, the duty cycle is increased to almost 100%. To improve the linear velocity range represented by OCTA signals, different inter-B-scan intervals (at least two) are required to visualize flow at different speeds. In our scanning protocol, the time between the first and second repetition is different than the time between second and third repetition, allowing a total of 3 different inter-scan times (1-2, 2-3 and 1-3) to be computed to improve flow quantification. A retinal OCTA of a healthy subject was acquired using our 400-kHz swept source OCT system. The volumetric scan was acquired in less than two seconds, potentially minimizing the prevalence of motion artifacts, which are more predominant in the scanning intervals most sensitive to slow speed flow. By averaging the three different images generated by 3 different inter-scan times, flow with large linear range (up to 5.2 mm/sec according to our prior calibration) is apparent on en face OCTA.

While the most common method used to evaluate and survey patients with Barrett’s Esophagus (BE) is endoscopic biopsy, this procedure is invasive, time-consuming, and suffers from sampling errors. Moreover, it requires patient sedation that increases cost and mandates its operation in specialized settings. Our lab has developed a new imaging tool termed tethered capsule endomicroscopy (TCE) that involves swallowing a tethered capsule which utilizes optical coherence tomography (OCT) to obtain three-dimensional microscopic (10µm) images of the entire esophageal wall as it traverses the luminal organ via peristalsis or is retrieved by pulling up tether. As opposed to endoscopy, TCE procedure is non-invasive, doesn’t require patient sedation and mitigates sampling error by evaluating the microscopic structure of the entire esophagus. The merits of TCE make it a suitable device to investigate the microscopic natural history of BE in a longitudinal manner.
Here, we present our initial experience of a multicenter (5-site) clinical trial to study the microscopic natural history of BE. The TCE device used for the study is the new generation capsule with the ball lens optical configuration and a distal scan stepper motor, which provides 30µm (lateral) resolution and 40Hz imaging rate. The portable OCT imaging system is a custom in-house built swept source system and provides 7µm (axial) at a 100 kHz A-line rate with a center wavelength of ~1310 nm. To date, we have successfully enrolled 69 subjects at all sites (MGH: 33, Columbia University: 11, Kansas City VA: 10, Mayo Jacksonville: 8, Mayo Rochester: 7) and 59 have swallowed the capsule (85.5%). There have been no reported adverse events associated with TCE procedure. High-quality OCT images were reliably obtained from patients who swallowed the device, and BE tissues were identified by expert readers. Our initial experience with TCE in a multicenter study demonstrates that this technology is easy to use and efficient in multiple clinical settings. Completion of this longitudinal study is likely to provide new insights on the temporal progression of BE that may impact management strategies.

We present an improved time-domain optical coherence tomography technique designed for ultrahigh-resolution B-scan imaging in real-time. The technique, called line-field confocal optical coherence tomography, is based on a Linnik-type interference microscope with line illumination using a supercontinuum laser and line detection using a line-scan camera. Bscan imaging with dynamic focusing is achieved by acquiring multiple A-scans in parallel. In vivo cellular level resolution imaging of skin is demonstrated at 10 frame/s with a penetration depth of ∼ 500 μm, with a spatial resolution of 1.3 μm × 1.1 μm (transverse × axial).

Full-field swept-source optical coherence tomography (FF-SS-OCT) provides high-resolution depth-resolved images by parallel Fourier-domain interferometric detection. Traditionally, FF-SS-OCT suffers from the cross-talk-generated noise from spatially coherent lasers. This noise reduces the image quality and limits wide adaptation of FF-SS-OCT for practical and clinical applications. To tackle this problem, we demonstrate and implement the spatiotemporal optical coherence (STOC) manipulation. In STOC, the phase of light in one of the interferometer arm is modulated in time with inhomogeneous phase masks displayed sequentially on the SLM. This modulation is synchronized with light acquisition to effectively control the spatial coherence of the detected light. A term "effectively" means that we do not generate the secondary source with imposed coherence properties (e.g. spatial incoherence). Instead the idea is to tailor the incident light to constrict the region of high fringe visibility to the spatial extents individual detection channels. Hence, SLM pixels are grouped into small blocks of uniform phase shifts. Then, phases are varied in time to modulate the light incident on the sample. By matching the dimensions of the SLM blocks to spatial extents of detection channels, we can de-correlate light from each channel. The unwanted interference between channels is washed-out and the cross-talk-generated noise is suppressed, helping to improve image quality. Here, the STOC approach is validated by imaging 1951 USAF resolution test chart covered by diffuser, scattering phantom and the rat skin ex vivo. Our results show a promising enhancement of the FF-SS-OCT capabilities that can be beneficial for imaging biological samples.

We implemented a combined visible light optical coherence microscopy (OCM) and fluorescence imaging platform. A supercontinuum light source in combination with a variable filter box (NKT Photonics) provided a spectral range of 425-680 nm. The OCM setup consists of a Michelson interferometer and a custom made spectrometer. Specification measurements were performed and an axial resolution of 0.88 μm in brain tissue was achieved. The transversal resolution was dependent on the objective lenses and varied from 2 to 8 μm. To change from OCM to fluorescence imaging, two mirrors had to be simply flipped into the light beam in the setup. For acquisition of fluorescence images, a photon multiplier tube (PMT) was used to detect light which had passed through a matched combination of emission, dichroic and excitation filters. As a first proof of concept, a fluorescence phantom consisting of curcumin powder mixed with mounting medium was imaged. The OCM images showed the three-dimensional structure of this phantom and specific contrast was gained by fluorescence imaging. As a control case, mounting medium without curcumin powder was imaged and no fluorescence was observed. One hallmark of Alzheimer's disease (AD) is the development of extracellular amyloid-beta plaques in the brain. The three-dimensional structure of these plaques was investigated with micrometer scale resolution using the OCM system. Curcumin can be used to specifically label amyloid-beta deposits. Curcumin stained brain slices of an AD mouse model were imaged and a specific contrast was gained by the fluorescence.

Quantitative measurements of lung microvessels would benefit characterization of vascular function and remodeling in pulmonary vascular diseases. Previous studies have evaluated the utility of micro-CT in conjunction with exogenous radiopaque silicone polymer injection (Microfil) to visualize vascular networks in whole organs. However, micro-CT resolutions are limited and Microfil perfusion may lead to incomplete vessel filling and vessel rupture. Optical coherence microscopy (OCM) enables depth-resolved volumetric imaging of tissue scattering with micron isotropic resolution and may be an alternative to micro-CT. Here, we present a novel method for quantitative measurements of lung vasculature using multi-volumetric OCM. Murine lungs were perfused with scattering contrast, fixed, and optically cleared. The lungs were then imaged using a custom-built OCM system with overlapping volumetric datasets and mosaicked in post-processing. OCM data was collected on a custom-built SD-OCT system and integrated with a control system to synchronize OCM data acquisition/archiving with three-axis motorized stages for multi-volumetric mosaicking. A Bessel illumination scheme was used to extend the Rayleigh range and depth-of-field by ~40% while maintaining high lateral resolution. A cleared lung lobe was imaged with 840 OCM volumes (7x12x10) that were acquired over an 8x13x1.43 mm slab with ~2 μm isotropic resolution. The resulting data was segmented in post-processing to quantify vessel diameters. We believe this proof-of-concept demonstrates the utility of our OCM and tissue preparation approach, which can be extended to compare microvasculature changes in entire lung lobes in animal models of pulmonary disease.

Polarization sensitive optical coherence tomography (PS-OCT) is a functional extension of optical coherence tomography (OCT). It provides addition information of the sample based on by analyzing polarization states of the backscattering light. Serval PS-OCT such as free-space optics, single model fiber and polarization maintaining (PM) fibers systems have been developed so far. However, the free space PS-OCT is prone to systematic errors and impractical in clinic settings. Dues to the uncontrolled polarization states, single model-based optics requires additional compensation and self-calibration techniques are required. Traditional PM fiber preserves the polarization states but found expensive and can not be used in rotating endoscopic or catheter probe. In this study, we develop a novel scheme of PS-OCT implementation using specific PM fiber, known as spun fiber, which has a structure of PM fiber twisted along the fiber optic axis and distinguishes two circular opalization states with different propagation speeds. Spun fiber has the advantages in maintaining the polarization states and regardless of fiber bends. The orientation insensitivity of the spun fiber makes it of great potential in endoscopic PS-OCT system. We tested our spun fiber-based PS-OCT system on chicken breast sample. The phase retardation image shows clear muscle structures compared to intensity-based OCT images, indicating our PS-OCT system has the ability to detect tissue birefringence.

Reconstruction of an OCT tomogram assumes that the signal originates from singly backscattered light. Light that has been multiply scattered only reduces image contrast and resolution. It has been demonstrated that multiple scattering impacts the OCT signal, especially in case of relatively large spherical particles with a size exceeding the wavelength used for OCT. Here, we investigate the association of multiple scattering with depolarization, as measured with polarization sensitive OCT, to verify if it can explain the depolarization previously observed in lipid-rich atherosclerotic plaques imaged with intravascular polarimetry. Atheromatous plaques consist primarily of macrophage cells that have ingested substantial amounts of lipids, stored as intracellular lipid droplets that increase in size as the plaque progresses. Excessive concentration of cholesterol leads to the nucleation of small cholesterol crystals. The strong birefringence and random orientation of these crystalline particles may contribute to the observed depolarization. However, detection of light multiply scattered by spherical particles may better explain the observed strong depolarization. Using the extended Huygens Fresnel principle we estimate the ratio between the contribution of singly and multiply scattered light to the OCT signal of aqueous microsphere suspensions, and compare it to the experimentally observed depolarization signal. Understanding the mechanism of depolarization seen in liquid-rich plaques may offer insight into the size and concentration of the lipid particles, which would be diagnostically relevant.

The feasibility of using optical coherence tomography, a label-free, non-invasive technique, to monitor three-dimensional (3D) morphology and pathology of tumor spheroids has been previously demonstrated. Growth kinetics of each spheroid, with its size and volume measured, could be accurately characterized. However, the previous system was not fully optimized for the collection of spheroid data from the whole plate. Here, in a follow-up study, we demonstrated a high-throughput optical coherence tomography (HT-OCT) platform capable of performing automatic 3D imaging and analyses for all tumor spheroids in a multi-well plate. The total screening time for a 96-well plate was ~23 min, including the OCT acquisition time of ~3.2min. Employing HT-OCT system, we successfully characterized a plate of tumor spheroids modeling cell invasions, with 3 different drug treatments. The HT-OCT system can be a powerful tool for fast, robust 3D morphological characterization of simple and complex spheroids for different cancer models. Further, they can also be utilized to analyze other models like organoids and artificial skins.

We report realizations of OCT combining conventional structural imaging, polarization-sensitive one, as well as allowing for real-time angiographic, elastographic and lymphangiographic modalities with manual-operation capabilities. Among the main features of the developed device one can point out on-flight imaging of microvascular network with feedback for clinicians when performing angiography; in elastography - robust "vector" method of interframe phasevariation gradient estimation and stiffness quantification using reference silicone layers; lymphangiography utilizing pixel statistics beyond conventional amplitude thresholding, etc. These capabilities are ensured by the developed optical schemes of the probe, signal receiving parts, as well as computationally efficient signal processing methods. Examples of the developed device usage in preclinical and clinical applications are discussed (efficient criteria for PDT success; angiographic monitoring of complications during radiotherapy; elastographic classification of tumor and non-tumor regions; detailed imaging of fairly rapid transient and slowly varying deformations in laser-assisted reshaping of collagenous tissues; lymphangiography-based diagnostics in gynecology; otolaryngologic applications for diagnosing inner ear diseases, etc.)

Approximately a quarter of patients undergoing breast conserving surgery will need further surgery as close or involved surgical margins suggest they may have residual tumour in the breast. Handheld imaging probes capable of scanning the surgical cavity during the surgery have the potential to improve intraoperative assessment of surgical margins in breast conserving surgery thus allow real time assessment of completeness of tumour excision. In this paper, we present a handheld optical coherence elastography (OCE) probe, allowing us to acquire a 3D quantitative elastogram of a 6×6×1.5 mm volume in 3.4 seconds. Our technique is based on a compression OCE technique, referred to as quantitative micro-elastography (QME), where a compliant silicone layer is incorporated to measure stress at the tissue surface. To perform handheld scanning, we implemented a rapid scan pattern to enable B-scan rates of 215 Hz using a microelectromechanical system (MEMS) scanner: minimizing the time difference between B-scan pairs used to generate displacement maps thus minimizing the motion artefact caused by hand motion. We present handheld scans acquired from silicone phantoms where the motion artefact is barely noticeable. In addition, freshly dissected human breast tissue from a mastectomy was scanned with the handheld probe. The breast tissue elastograms are validated using standard histology and demonstrate our ability to distinguish stiff regions of tumour from benign tissue using this probe.

Optical coherence tomography (OCT) allows for non-contact, high resolution, volumetric imaging of biological tissue and has become an indispensable ophthalmic imaging technique. However, conventional, commercial OCT systems require a cooperative, sitting patient typically stabilized by a head and/or chin rests. Additionally, current clinical systems are designed for imaging either the anterior or posterior segment of the eye exclusively. While these limitations are not severe in the ophthalmic clinic, they do limit the use of OCT in other more challenging medical environments where novel “whole eye” imaging could provide value, such as in the military theater or emergency department (ED). One solution to eliminate the need for a patient to sit upright and be stabilized during imaging would be a hand-held probe positioned and stabilized by the photographer or physician. Here we describe a hand-held OCT probe for simultaneous imaging of the anterior chamber (13.3 mm diameter field-of-view) and posterior segment (40° as measured from the pupil nodal point) simultaneously. The use of polarization multiplexing allows for two independent imaging channels which enable a wide posterior segment field-of-view and the ability to control the posterior segment path length and focal depth independently from the anterior chamber channel. Additionally, the probe was designed for a relatively compact form factor.

Investigations on retinal vasculature and blood flow are of interest for understanding and diagnostics of numerous ocular diseases. Conventional OCT systems use various scan patterns like linear B-scans, circular scans around the optic nerve head, or raster scans for 3D data acquisition. However, for some studies it is preferable to have customized scan patterns that can, e.g., follow the trace of an arbitrary linear structure in the retina, such as a vessel. In this work, we present an OCT instrument with an integrated retinal tracker that allows repeated scans along an arbitrary trace, whereby ocular motions are corrected by the retinal tracker. The setup comprises an OCT system and a line scanning laser ophthalmoscope (LSLO). The OCT subsystem operates at a center wavelength of 860 nm, with a bandwidth of 60 nm and an A-scan rate of 70kHz. The LSLO system operates at 790 nm and at a frame rate of 60 Hz. This system was used for reflectivity and Doppler imaging along retinal vessels. In a first step, the vessel is manually marked on the LSLO image. Then, repeated scans along the vessel trace are performed (2048 A-scans per scan along trace, up to 500 scans along the trace). The intensity images show a clear delineation of the vessel walls, the phase difference (Doppler) tomograms allow for a time-resolved analysis of blood flow along the vessel over the cardiac cycle.

High quality visualization of the retinal microvasculature can improve our understanding of the onset and development of retinal vascular diseases, especially Diabetic Retinopathy (DR), which is a major cause of visual morbidity and is increasing in prevalence. Optical Coherence Tomography Angiography (OCT-A) images are acquired over multiple seconds and are particularly susceptible to motion artifacts, which are more prevalent when imaging individuals with DR whose ability to fixate is limited due to deteriorating vision. The sequential acquisition and averaging of multiple OCT-A images can be performed for removing motion artifact and increasing the contrast of the vascular network. As motion artifacts often irreversibly corrupt OCT-A images of DR eyes, a robust registration pipeline is needed before feature preserving image averaging can be performed.
In this report we present an improvement upon a novel method for the acquisition, processing, segmentation, registration, and averaging of sequentially acquired OCT-A images, to correct for motion artifacts in images of DR eyes. Image discontinuities caused by rapid micro-saccadic movements and image warping due to smoother reflex movements were corrected by strip-wise affine registration and subsequent local similarity-based non-rigid registration. Where our previous work was limited by the need for at least one image containing no motion artifact, thus reducing its clinical relevance, this novel template-less method stitches together partial images to form complete, motion-free images. These techniques significantly improve image quality, increasing the value for clinical diagnosis and increasing the range of patients for whom high quality OCT-A images can be acquired.

A depth-multiplexed fiber-based PS-OCT system is used to extract local polarization information of retinas of age-related macular degeneration (AMD) patients in different stages. In the end stage of wet AMD, retinal structures are replaced with fibrous tissue which leads to irreversible loss of vision. Accurate imaging and evaluation of the lesions is important for reliable diagnosis and treatment of AMD. However, no imaging techniques exist which can clearly distinguish a fibrotic lesion from non-fibrotic neovascular tissue which is still active.
With PS-OCT fibrous tissue in the retina of AMD patients can be detected and quantified using its birefringent properties. Images from previous research often show cumulative phase retardation, where the polarization state of every pixel is compared to the polarization state at the surface of the retina. However, a quantity which is linearly related to the amount of birefringent tissue is more desirable for clinical interpretations. In the presented research, a new method is used to obtain depth-resolved local birefringence images which has only been used on breast tissue before. In this method, the birefringent quantities (linear phase retardation) are extracted from the differential Mueller matrix.
In the images of retinas from AMD patients, fibrotic lesion areas can be recognized and separated from non-fibrotic areas. An improvement to localize birefingence in depth while maintaining similar image quality is demonstrated. This provides new possibilities for clinical research to monitor the development of AMD and to assess the response to treatment.

Elastic light scattering spectroscopy (ELSS) has been proven as a powerful tool in characterizing tissue native structures with superb sensitivity. As a widely used technique, optical coherence tomography (OCT) would have been well suited for ELSS measurement by using a broadband light source. However, OCT-based ELSS is largely hampered by the limited k-space spectral bandwidth from all existing OCT systems. To overcome this barrier, we report a simple all fiber-based setup to implement dual-channel visible and near infrared (NIR) optical coherence tomography (vnOCT) for human retinal imaging, bridging over 300nm spectral gap. Remarkably, we discovered a newly available fiber that supports single-mode propagation and maintains high interference efficiency for both visible and NIR light with fringe visibility of 97% and 90%, respectively, which was previously considered impossible to use the same fiber components for such a broad range of wavelengths. Longitudinal chromatic aberration from the eye is corrected by a custom-designed achromatizing lens. As retinal imaging being an important OCT application, we demonstrated vnOCT on human retina and further developed robust ELSS analysis method to quantify spectroscopic contrast in several import layers of human retina. This vnOCT platform and method of ELSS analysis open new opportunities in understanding structure-function relationship in the human retina and in exploring new biomarkers for retinal diseases.

A new method is presented, called Spectral Contrast Optical Coherence Tomography, which utilizes the visible spectrum of blood instead of doppler or speckle contrast to locate blood vessels. This is seen as a significant improvement for OCT angiography, since sample motion no longer affects vessel contrast, repetitive scanning is not required, and non-flowing blood can be imaged. A visible Optical Coherence Tomography system from 500-700 nm was used and the differential spectral intensity of two short time Fourier Transform Kaiser sampling windows centered at 557 nm and 620 nm provided contrast revealing blood vessel location. This approach allows for single-scan endogenous contrast angiography all the way down to the capillary level. We demonstrate the method by imaging the vasculature of human oral mucosa and the lymphatics and vasculature of freshly sacrificed mouse tissue.

Endoscopic evaluation of the colorectum is limited to the mucosal surface and provides no functional or structural information regarding subsurface changes. Targeted diagnostics and individualized treatment, however, requires this information. In this ex vivo study of human colorectal tissue, we use swept-source optical coherence tomography to create quantified subsurface scattering coefficient maps of normal and cancerous tissue. Specifically, we use a novel wavelet-based-curve-fitting method to generate subsurface scattering coefficient maps. The angular spectra of scattering coefficient maps of normal tissues exhibit a spatial feature distinct from those of abnormal tissues. The en face scattering coefficient maps of the normal colon contain a large area of homogenous scattering coefficients with periodic dot patterns, while the scattering coefficient map of cancer region shows a large area of heterogeneous scattering coefficients. An angular spectrum index to quantify the differences between the normal, abnormal, and treated tissues is derived, and its strength in revealing subsurface cancer in ex vivo samples is statistically analyzed. Using this index, we differentiate malignant from normal colonic tissue. Additionally, we also found that rectal cancers completely destroyed by preoperative treatment appear much more similarly to normal tissue than the original malignancy. The study demonstrates that the angular spectrum of the scattering coefficient map can effectively reveal subsurface colorectal cancer and help clinicians identify patients who don’t require surgical intervention.

Optical coherence tomography angiography (OCTA) is an important tool for investigating vascular networks and microcirculation in living tissue. Traditional OCTA detects blood vessels via intravascular dynamic scattering signals derived from the movements of red blood cells (RBCs). However, the low hematocrit and long latency between RBCs in capillaries make these OCTA signals discontinuous, leading to incomplete mapping of the vascular networks. OCTA imaging of microvascular circulation is particularly challenging in tumors due to the abnormally slow blood flow in angiogenic tumor vessels and strong attenuation of light by tumor tissue. Here we demonstrate in vivo that gold nanoprisms (GNPRs) can be used as OCT contrast agents working in the second near infrared window, significantly enhancing the dynamic scattering signals in microvessels and improving the sensitivity of OCTA in skin tissue and melanoma tumors in live mice. This is the first demonstration that nanoparticle-based OCT contrast agent works in vivo in the second near infrared window, which allows deeper imaging depth by OCT. With GNPRs as contrast agents, the post-injection OCT angiograms showed 41% and 59% more microvasculature than pre-injection angiograms in healthy mouse skin and melanoma tumors, respectively. By enabling better characterization of microvascular circulation in vivo, GNPR-enhanced OCTA could lead to better understanding of vascular functions during pathological conditions, more accurate measurements of therapeutic response, and improved patient prognoses.

Intraoperative optical coherence tomography (iOCT) enables volumetric imaging of surgical maneuvers. While previous studies have demonstrated the utility of iOCT for verifying completion of surgical goals, images were acquired over static field-of-views (FOVs) or required manual tracking of regions-of-interest (ROIs). The lack of automated instrument-tracking remains a critical barrier to real-time surgical feedback and iOCT-guided surgery. Previously presented approaches to address this include active stereo-vision based instrument tracking, which was limited to imaging in the anterior segment; and instrument-tracking using volumetric OCT data, which was limited by OCT acquisition speeds and fundamental trade-offs between sampling density and FOV. We previously presented spectrally-encoded coherence tomography and reflectometry (SECTR), which provides simultaneous imaging of spatiotemporally coregistered orthogonal imaging planes (en face and cross-sectional) at several gigapixels-persecond. Here, we demonstrate automated surgical instrument-tracking and adaptive-sampling of OCT using a combination of deep-learning and SECTR. A GPU-accelerated deep neural network was trained using SER images for detection and localization of 25G internal limiting membrane (ILM) forceps at up to 50 Hz. Positional information was used for acquisition of adaptivelysampled SER frames and OCT volumes, which were densely-sampled at the instrument tip and sparsely-sampled elsewhere to retain tracking features over a large field-of-view. We believe this method overcomes critical barriers to clinical translation of iOCT and offers advantages over previous approaches by 1) reducing the instrument-tracking problem to 2D space, which is more efficient than 3D tracking or pose-estimation, and allows direct leveraging of the recent advances in computer-vision software and hardware; and 2) decoupling tracking speed and performance from OCT system and acquisition parameters.

The purpose of this study was to develop and evaluate the performance of a convolutional neural network (CNN) that uses a novel A-line based classification approach to detect cancer in OCT images of breast specimens. Deep learning algorithms have been developed for OCT ophthalmology applications using pixel-based classification approaches. In this study, a novel deep learning approach was developed that classifies OCT A-lines of breast tissue. De-identified human breast tissues from mastectomy and breast reduction specimens were excised from patients at Columbia University Medical Center. A total of 82 specimens from 49 patients were imaged with OCT, including both normal tissues and non-neoplastic tissues. The proposed algorithm utilized a hybrid 2D/1D convolutional neural network (CNN) to map each single B-scan to a 1D label vector, which were derived from manual annotation. Each A-line was labelled as one of the following tissue types: ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), adipose, and stroma. Five-fold cross-validation Dice scores across tissue types were: 0.82-0.95 for IDC, 0.54-0.75 for DCIS, 0.67-0.91 for adipose, and 0.61-0.86 for stroma. In a second experiment, IDC and DCIS were combined as a single tissue class (malignancy) while stroma and adipose were combined as a second tissue class (non-malignancy). In this setup, the experiment yielded five-fold cross-validation Dice scores between 0.89-0.93, respectively. Future work includes acquiring more patient samples and to compare the algorithm to previous works, including both deep learning and traditional automatic image processing methods for classification of breast tissue in OCT images.

Optical coherence tomography(OCT) imaging of bladder is gaining recognization due to the capability of noninvasive cross-sectional imaging of the bladder at the micron-level resolution and a relatively large field of view. Previous studies have shown the potential of OCT image to enhance detection of bladder transitional cell carcinoma(TCC). However, quantitative OCT image analysis for affirmative identification of bladder tumor remains a challenge[1]. Here, we report a novel method to enhance detection of TCC based on OCT images by analyzing anatomical and textural alteration of bladder. Specifically, OCT images are first processed with Dual Tree Complex Wavelet Transform denoising algorithms to reduce image speckle noise. Then, the layer segmentation method that mainly based on a dual path graph searching algorithm is performed on the denoised images to delineate three layers of bladder. The segmentation results show improved effectiveness and robustness in comparison to conventional graph theory based method. With layer segmentation, multiple measurements including layer thickness and texture can be quantified. The significant difference in quantified metrics between TCC and normal bladder indicate the potential use of those metrics for TCC identification. The proposed method provides valuable insights into TCC and has the potential to enhance the detection of tumor in the clinic.

Optical coherence tomography angiography (OCTA) is a promising tool for imaging subsurface microvascular networks owing to its micron-level resolution and high sensitivity. However, it is not uncommon that OCTA imaging tend to suffer from strip artifacts induced by tissue motion. Although various algorithms for motion correction have been reported, a method that enables motion correction on a single en face OCTA image remains a challenge. In this study, we proposed a novel motion correction approach based on microvasculature detection and broken gap filling. Unlike previous methods using registration to restore disturbed vasculature during motion artifact removal, tensor voting is performed in individual projected image to connect the broken vasculature. Both simulation and in vivo 3D OCTA imaging of mouse bladder are performed to validate the effectiveness of this method. A comparison of in vivo images before and after motion correction shows that our method effectively corrects tissue motion artifacts while preserving continuity of vasculature network. Furthermore, in vivo results of this technique are presented to demonstrate the utility for imaging tumor angiogenesis in the mouse bladder.

A polarization sensitive extension was implemented into our bright and dark field optical coherence tomography (BRAD-OCT) system. The few-mode fiber detection enables the analysis of the scattering profiles of biological tissues by sensing the sample backscattering information at larger angles through the different fiber modes, enabling simultaneous detection of the bright and dark field. Since the polarization state of light scattered at different angles strongly depends on the tissue microstructure, the polarization sensitive detection improves the imaging capabilities of BRAD-OCT. The system performance was evaluated in phantoms of differently sized microparticles, showing different polarization characteristics for the different fiber modes. Preliminary analysis has been performed in ex vivo brain tissue, where stokes vectors analysis of the different modal images reconstructed by our BRAD-OCT setup indicates that polarization sensitive in combination with BRAD detection is a promising tool for investigating the scattering properties of biological tissues.

Over the past decades, optical coherence tomography has emerged as an important imaging technique to study biological processes through its ability to perform three-dimensional imaging at high acquisition rates and non-invasively. Furthermore, OCT has shown a growing interest in brain imaging through its capacity in obtaining functional information such as cellular viability, hematocrit and blood flow velocity.
Although OCT can reach image depths spanning a few millimeters, the effective imaging depth is typically dictated by the depth-of-field of the imaging optics. In traditional OCT systems, this depth-of-field is given by the Rayleigh range and is thus coupled to the lateral resolution. As such, increasing the numerical aperture of the system reduces the imaging depth, ultimately hampering the depth-multiplexing advantage of OCT. Wavefront engineering schemes have been devised to overcome this limitation, providing the OCT systems with an extended-focus. We present here two extended-focus OCT systems (xf-OCT) optimized for cerebral imaging. The first system operates in the visible wavelength range and is designed to image the superficial cortex of mice at high contrast and at high resolution. Its high axial and lateral resolution of 0.8 and 1.4 um respectively, maintained over 200 um enable resolving structures such as myelinated axons, neuronal cells and micro-vessels in vivo. The second system is optimized for deep microvascular cortical imaging and operates in the infrared spectral range. Through its extended-focus and increased penetration, the second system can provide maps of cortical microvasculature over 800 um in depth in the cortex in vivo.

The penetration depth of optical microscopy in biological tissue is limited by attenuation due to absorption and scattering. Scattering decreases with wavelength, while water absorption is locally minimized at so-called infrared “optical windows.” Of the four infrared optical windows, light in the longest wavelength window at 2200 nm experiences the least scattering and the most water absorption. Therefore, the 2200 nm window is rarely used for biological microscopy. However, fractional water content differs greatly between tissues. Therefore, the best optical window for deep imaging may depend on tissue type. Here, we demonstrate the benefits of the 2200 nm optical window for imaging through the skull, which is highly turbid with a relatively low water content. A spectral domain optical coherence tomography (OCT) system at ~2200 nm was built. A maximum sensitivity of 86 dB and a tissue axial resolution of 13.9 µm were achieved. To assess relative contributions of scattering and water absorption, 2200 nm was compared with 1300 nm. In vivo cortical vasculature was imaged angiographically through the intact skull in mice. Overall 2200 nm experienced less attenuation through the intact skull. In cortical layer I, 1300 nm, which experiences more scattering but less water absorption, and 2200 nm, which experiences less scattering but more water absorption, exhibit similar attenuation. In deeper cortical layer II/III, higher attenuation was observed at 2200 nm due to higher water absorption. Thus the infrared window at 2200 nm may provide advantages for imaging layers at or near the cortical surface through thick skull.

Prenatal substance abuse is a major public health concern. Much research has been focused on alcohol and other drug use, but there is a lack of information about prenatal cannabinoid use. Nevertheless, marijuana use during pregnancy increases the risk of a stillbirth by approximately 2.3X. Synthetic cannabinoids (SCB) are a group of heterogeneous compounds which were developed to understand the endogenous cannabinoid system and as potential therapeutics. SCBs are legally available for purchase in several places, and the use of natural and synthetic cannabinoids is high among women of reproductive age. Combined with the prevalence of unplanned pregnancies, the high use of cannabinoids may lead to an increase in prenatal exposure to cannabinoids. Early studies have shown morphological and behavioral anomalies similar to fetal alcohol syndrome. Even though the mechanisms of Δ9 -tetrahydrocannabinol (Δ9 -THC), the major psychoactive component of marijuana, and SCB are similar, there are several important differences. Subsequently, some SCBs have a 40 to 600 fold higher potency than Δ9 -THC. However, there is paucity of research focused on the prenatal effects of SCBs. This study uses correlation mapping optical coherence tomography (cm-OCT) to evaluate acute changes in the murine fetal brain vasculature in utero after exposure to CP-55,940, a well-characterized and commonly used reference compound in cannabinoid research. Our results showed a rapid decrease in parameters quantifying vasculature, i.e., vessel area density, and vessel length fraction, as compared to the sham group, demonstrating a dramatic and rapid effect of cannabinoids on fetal brain vasculature. Our work shows the need for further research on the effects of cannabinoids on fetal development.

Objective optical assessment of photoreceptor function may permit earlier diagnosis of retinal disease than current methods such as perimetry, electrophysiology, and clinical imaging. Recent work with adaptive optics (AO) flood imaging, conventional OCT and phase-sensitive full-field OCT have revealed apparent changes in photoreceptor outer segment (OS) length in response to visible stimuli. In this work, we describe an AO-OCT system designed to measure these stimulus-evoked OS length changes. The OCT subsystem consisted of a Fourier-domain mode-locked laser that acquires A-scans at 1.64MHz and an AO subsystem providing diffraction-limited imaging with a closed-loop correction rate of 20Hz. To our knowledge this is the highest-speed AO-OCT system developed to date. Visible stimuli were delivered using a LED-based Maxwellian view channel incorporated into the system. In a dark-adapted healthy subject, 1-deg square volumetric images were acquired at a rate of 32Hz. Images were acquired for 10s, with a 10ms bleaching stimulus flash with variable intensity. Strip-based registration was used to track individual cones in the volume series, and time series of the resulting depth-resolved complex signal were analyzed. Stimulus-evoked changes in the morphology of OS and RPE were observed in the M-scan amplitude. In the M-scan phase, the difference between the IS/OS and COST was shown to increase in response to the stimulus flash, and the magnitude of the phase change depended upon flash intensity. These results suggest that cone OS elongates in response to visible stimuli, and that the length change scales with stimulus intensity.

Aberration-corrected imaging of human photoreceptor cells, whether hardware or software based, presently requires a complex and often expensive setup. Here, we demonstrate a simple and inexpensive off-axis full-field time-domain optical coherence tomography approach to acquire volumetric data of in vivo human retina. Full volumetric, laterally phase stable data are recorded. The lateral phase stability allows computational aberration correction, which enables us to visualize single photoreceptor cells. In addition, our approach is able to correct large aberrations and is thus feasible for the numerical correction of ametropia in post processing. Our implementation of full-field OCT combines a low technical complexity with the possibility to use the phase of the recorded light for computational image correction.

In this study we describe our novel Multi-Scale and multi-Mode Sensorless Adaptive Optics OCT system (MSM-SAO-OCT). Our system expands upon our previously reported work by introducing a zoomable collimator, phase calibration interferometer, and polarization diversity detection module. By using a zoomable collimator into the system setup, we allow an adjustable probing beam diameter without the need to change the optical setup, permitting imaging with both low and high lateral resolution (18 µm – 6 µm) at various Fields of View (FOV) within diffraction limited resolution. By employing SAO optimization algorithm, different morphological structures and microvasculature in a retina were clearly visualized after wavefront aberration correction with dual deformable optical elements – Variable Focus Lens (VFL) for defocus and a Multi-Actuator Adaptive Lens (MAL) for two astigmatisms. For retinal vasculature imaging, MSM-SAO-OCT system generates flow-specific contrast as measuring amplitude of complex variance from the multiple OCT B-scans from the same transverse location after stabilizing OCT signals in a phase using a static interference signal from phase calibration interferometer. In addition, the use of polarization diversity detection allows to create Degree Of Polarization Uniformity (DOPU) contrast using for visualization of the Retinal Pigment Epithelium (RPE) with its inherent tissue characteristic (polarization scrambling). In order to demonstrate functionality and clinical utility of the MSM-SAO-OCT system, in vivo human retinal imaging was performed on research subjects, and imaging results are presented and discussed.

Full-Field Optical Coherence Tomography (FF-OCT) offers aberration independent resolution. This inherent property makes FF-OCT a promising imaging modality for 3D high-resolution retinal imaging. Nevertheless, ocular aberrations affect signal reduction, imposing Adaptive Optics. Here we investigate the best strategy to compensate for ocular aberrations in our FF-OCT setup, in terms of wavefront measurement and correction. The use of wavefront sensorless approach based on the FF-OCT signal level is investigated. Moreover, a strategy of static wavefront correction in a non-conjugated pupil plane and next to the eye’s pupil, just like spectacles, favoring a compact and non-complex AO design, is also investigated. Additionally, the use of wavefront corrector devices such as an adaptive liquid lens (correcting to defocus and astigmatism) and multi-actuator adaptive lenses (correcting up to the 4th order Zernike polynomial) are evaluated. Finally, we expect the implementation of one or a combination of the studied strategies into our FF-OCT setup to lead to the first in-vivo retinal images obtained using AO-assisted FF-OCT, for different retinal layers with enhanced SNR and a 3D high-resolution.

Optical coherence microscopy (OCM) provides non-invasive, label-free, cellular-resolution imaging based on optical scattering contrast. Its interferometric detection captures the optical field, providing opportunities for computational reconstruction. However, the depth coverage of OCM is restricted by defocus and photon collection, and its penetration depth is limited by multiple scattering (MS). Here, we propose integrating hardware and computational adaptive optics in different ways, to improve the throughput, penetration depth, and contrast of volumetric OCM. This hybrid adaptive optics (hyAO) approach splits the image formation process into a combination of hardware and computation components. For sparse sample imaging, we generated astigmatism using hardware adaptive optics (HAO) to achieve a more equalized photon distribution across depth, and removed the applied aberration (and defocus) via computational adaptive optics (CAO). We applied this hyAO method to perform 3D time-lapse imaging of in vitro fibroblast cell dynamics over a 1mm×1mm×1mm field-of-view with 2μm isotropic spatial resolution and 3-minute temporal resolution. The hyAO approach is not only beneficial for high-throughput volumetric imaging, but is also capable of suppressing MS/speckle. For scattering sample imaging, HAO was used to illuminate the sample volume with diverse aberrated point spread functions to decorrelate the MS/speckle fields, and CAO was applied to computationally mitigate the resolution penalty of these intentionally induced aberrations. By imaging with this aberration-diverse OCT using 12 volumetric reconstructions, we achieved a 10 dB enhancement in signal-to-background ratio at a USAF target plane beneath a scattering layer (7.2 scattering mean-free-path), and a 3× speckle contrast reduction within the scattering layer.

Speckle in optical coherence tomography (OCT) is a consequence of coherent detection scheme and is often considered as a noise submerging the micro-structures of biological tissue. In this work we present a novel method to suppress the speckle in OCT by introducing random phase shifts using a fully controlled segmented deformable mirror (DM) conjugated with imaging system pupil plane, allowing dynamic control of Point Spread Function (PSF) in the sample. These PSF modulations allow different set of scatters contribute to generation of un-correlated speckles, allowing for efficient suppression of speckle contrast by averaging multiple images. The speckle contrast suppression by the random shapes of deformable mirror was investigated in detail. We further present that the degradation of image intensity and resolution can be mitigated by using only the selected mirror shapes that correspond to the brighter OCT images, while maintains similar speckle suppression effect. Finally, the in vivo mouse retina imaging results demonstrate the capabilities of our method to enhance the visibility of subcellular micro-structures previously hidden behind the speckles.

The dynamic properties of subcellular organism are important biomarkers of health. Imaging subcellular level dynamics provides effective solutions for evaluating cell metabolism, and moreover, testing the responses of cells to pathogens and drugs in pharmaceutical engineering. In this paper, we demonstrate an innovative approach to contrast the subcellular motions by using eigen decomposition (ED) based variance analysis of time-dependent complex optical coherence tomography (OCT) signals. This method reveals superior contrast to noise advantage compared with intensity-based dynamic imaging regime. Further validation experiments were performed with B-mode imaging sections crossing a wide range of sampling frequencies, and on the patterned samples of yeast powder mixed with gelatin/TiO2-water solution. In addition, the proposed method was further used to image mouse cerebral cortex in vivo, suggesting the promising of ED based correlation power mapping in analyzing coupled dynamics of neuron activity and cerebral blood flow. The proposed technique promises efficient measurement of subcellular motions with high sensitivity and low artifact involvement, suggesting high potential for in vivo and in situ applications.

Variations in the mechanical properties of the extracellular environment can alter important aspects of cell function such as proliferation, migration, differentiation and survival. However, many of the techniques available to study these effects lack the ability to characterise cell-to-cell and cell-to-environment interactions on the microscopic scale in three dimensions (3D). Quantitative micro-elastography (QME) is an extension of compression optical coherence elastography that utilizes a compliant layer with known mechanical properties to estimate the axial stress at the tissue surface, which combined with axial strain, is used to map the 3D microscale elasticity of tissue into an image. Despite being based on OCT, limitations in post-processing techniques used to determine axial strain prevented QME to quantify the elasticity of individual cells. In this study we extend the capability of QME to present, to the best of our knowledge, the first images of the elasticity of cells and their environment in 3D over millimeter field-of-views. We improve the accuracy and resolution of QME by incorporating an efficient, iterative solution to the inverse elasticity problem using adjoint elasticity equations to enable QME to visualize individual cells for the first time. We present images of human stem cells embedded in soft gelatin methacryloyl (GelMa) hydrogels and demonstrate these cells elevate the stiffness of the GelMa from 3-kPa to approximately 25-kPa. Our QME system is developed using commercially available components that can be readily made available to biologists, highlighting the potential for QME to emerge as an important tool in the field of mechanobiology.

A leading cause of death and decrease in the quality of life in the USA is myocardial infarction (MI). Molecular and genetic analyses have revealed a plethora of information about critical processes involved with MI. However, there is a lack of accompanying information about cardiac tissue biomechanical properties, which may provide additional critical information to understand the tissue remodeling process after MI. In this work, we utilize two complementary noncontact optical coherence elastography (OCE) techniques to assess the changes in mouse cardiac tissue biomechanical properties 6 weeks after the MI was induced. A focused micro air-pulse induced localized displacements, which were detected by a phase-sensitive OCE (PhS-OCE) system. The localized tissue displacement was modeled by a spring-mass damper model to quantify the tissue stiffness. Additionally, the propagation of an air-pulse induced elastic wave was measured at various meridional angles as a complementary measurement of tissue stiffness and anisotropy. The damping results show that the MI caused a decrease in the stiffness of the cardiac tissue. Similarly, the analysis of elastic wave propagation showed that the cardiac tissue became softer and more isotropic after MI. These results show that OCE can detect the changes in cardiac tissue biomechanical properties after MI. OCE may also be useful for developing targeted therapies by identifying regions of cardiac tissue affected by MI.

The absorption of nanosecond laser pulses induces rapid thermo-elastic deformation in tissue. A sub-micrometer scale displacement occurs within a few microseconds after the pulse arrival. We investigate the thermo-elastic deformation using a 1.5 MHz phase-sensitive optical coherence tomography (OCT) system. An analysis of the results shows that the displacement is dominated by the optical absorption. By tuning the excitation wavelength, thermo-elastic displacement spectrum can be extracted, showing the similar features as optical absorption spectrum. By choosing proper excitation wavelength, targeted tissue type can be highlighted, which further enables a new imaging modality, so called thermo-elastic OCT.

Wave-based optical coherence elastography (OCE) is a rapidly emerging technique for localized elasticity assessment of tissues due to its high displacement sensitivity and simple implementation. This method does not require prior knowledge of mechanical load characteristics, such as the applied preload and applied stress on the sample. Currently, noncontact wave excitation has been accomplished with various methods, such as focused micro air-pulse and acoustic techniques. However, they are limited by the inability to target specific tissues and usually only image the transversely propagating elastic wave, which generally requires scanning the probe beam across the sample. In addition, the upper frequency components of the elastic waves are limited to a few kilohertz, which are sensitive to boundary conditions due to their long wavelengths. In this study, we demonstrated that rapid vaporization of perfluorocarbon inside dye nanoparticles that was excited by a pulsed laser excitation, termed “nanobombs”, can produce high frequency longitudinal elastic waves in tissue mimicking phantoms. The nanoparticles were excited by a 1064 nm pulsed laser, which was co-focused with the OCT probe beam. The longitudinal elastic waves, which propagated axially (i.e., following the optical path), were directly imaged by a phase-sensitive Fourier domain mode-locked based OCT system. The detected elasticity was validated with well-established air-pulse OCE and the “gold standard” uniaxial mechanical testing. The results demonstrate the feasibility of performing nanobomb elastography in tissue with the potential for targeting specific tissues and producing longitudinal elastic waves with high frequency content.

Fiber-based polarization-sensitive optical coherence tomography (PS-OCT) can measure cumulative Jones matrix that includes both the fiber-optic components of the interferometer and the sample. To derive a relative optic axis of the sample, a relative angle of the eigenpolarizations is often calculated on the Poincaré sphere. Here, we suggest a new approach using Jones formalism. This method is demonstrated for optic axis imaging of the inner retina and sclera in the posterior eye segment using PS-OCT with parallel detection of Jones-matrix elements.

We present a modified Fast Phase Unwrapping (FPU) algorithm and its application for calculation of the axial flow velocity and volumetric flow rate in Doppler optical coherence tomography (DOCT). We outline the FPU method and show that it can be implemented in Fourier-domain optical coherence tomography using Fourier transformations (4FT). We present two-dimensional (2D) and three-dimensional (3D) realizations of the algorithm to reconstruct unwrapped phase in numerical simulations, as well as in data collected from phantom. We demonstrate that the phase unwrapping outcomes of the 2D and 3D 4FT FPU algorithms depend on the phase noise in the input data. For low phase noise data both algorithms generate reliable results. With increasing noise, the 2D algorithm starts generating phase unwrapping errors earlier than the 3D version. With the phase noise larger than a limiting value, none of the algorithms provides error-free results. We demonstrate that within their phase noise applicability limits, the phase unwrapping methods enable calculation of volumetric flow rates in the flow phantom even in the presence of phase wraps. We demonstrate that application of phase unwrapping methods enables extension of the measurable flow velocities beyond the phase range limitation of the Doppler OCT data.

Doppler optical coherence tomography (DOCT) is a promising functional imaging modality for quantitative blood flow measurement. A major limitation of DOCT is that only the axial component of flow can be measured, which is strongly influenced by the geometry of the vasculature. To overcome this challenge, we proposed a new method to retrieve absolute blood flow velocity of the vascular networks with fully restored topology. The application of open active snake can detect the skeleton of vasculature network without the need of vasculature segmentation, allowing Doppler angle to be calculated. The discontinuity of vasculature induced by Doppler angle and the limited dynamic range is corrected by a tensor voting based method, enabling the measurement of absolute blood flow velocity along each fully connected vessel branch. We present the results of in vivo cerebral blood flow (CBF) networks to demonstrate the efficacy of the proposed method.

The calibration of a spectrometer is a critical step to obtain high-quality images in SDOCT. The spectra acquired with the spectrometer of an SDOCT are usually linear in wavelength, which should be linearly resampled in wavenumber before Fourier transform. Then the depth axis along each A-scan can be marked based on the wavenumber resolution of the spectrometer. In our project, we propose a novel method to calibrate the spectrometer. An easy and cost-effective way to generate Doppler frequency shift is introduced. Compared with previous methods, our method does not need additional hardware, such as a piezo shifter or a calibration light source. We prove that this Doppler frequency shift can be used to linearize the spectra from the wavelength domain to wavenumber domain. In addition, experiments prove that tissue images can be directly used for calibration without requiring mirror images. In other words, the calibration can be accomplished in situ, which is very useful for some clinical applications as the images can be calibrated by itself during or after imaging without relying on accurate manual calibration before imaging. For retinal imaging, this method may avoid spectrometer calibration using a model eyeball in the sample arm. In the further step, to mark depth axis, an interference signal is generated by coverslip which can provide maximum probing depth.

In this work, a Bessel beam from a deep seated negative axicon (DSNA) tip is utilized as a probe in the common-path interferometric configuration for cross-section tissue imaging. The DSNA is fabricated at the one end of an optical fiber by capillary action using chemical etching in hydrofluoric acid (48% HF) which generates high-quality Bessel beam. It has a small central spot size and large depth of field which ensure the quality of Bessel beam. This Bessel beam is used to probe the sample. The beam reflected back from the sample and couples with the probe treated as a sample beam which interferes with the reference beam generated at the air-axicon interface and the interference spectrum is acquired at detector end. This spectrum is further processed to obtain an image of the sample. The lateral and axial resolution of the system is ~3.3μm and ~6.9μm respectively. The experiments have been conducted on the tissue of the chicken muscle-fiber and heart. This optical fiber probe can be an ideal choice for an endoscopic probe in future.

Angular compounding is a technique for reducing speckle noise in optical coherence tomography that is claimed to significantly improve the signal-to-noise ratio of images without impairing their spatial resolution. Here we examine how focal point movements caused by optical aberrations in an angular compounding system may produce unintended spatial averaging and concomitant loss of spatial resolution. Experimentally, we accounted for such aberrations by aligning our system and measuring distortions in images, and found that when the distortions were corrected the speckle reduction by angular compounding was limited. Our theoretical analysis using Monte Carlo simulations indicates that “pure” angular compounding (i.e., with no spatial averaging) over our full numerical aperture (13° in air) can improve the signal-to-noise ratio by no more than a factor of 1.3. Illuminating only a partial aperture cannot improve this factor compared to a spatial averaging system with equivalent loss of resolution. We conclude that speckle reduction using angular compounding is equivalent to spatial averaging. Nonetheless, angular compounding may be useful for improving images in applications where depth of field is important. The distortions tend to be greatest off the focal plane, so angular compounding combined with our correction technique can reduce speckle with a minimal loss of resolution across a large depth of field.

We report on the use of the Complex Master-Slave (CMS) method to obtain a long axial range in a sweptsource OCT system, well above the axial range limit imposed by the k-clock of the optical source. This is achieved without the need for software-based k-domain re-sampling or employing an additional Mach-Zehnder interferometer providing a stable k-clock signal to the digitizer board. An imaging range of over 17 mm is reported in each case using a commercially available swept source from either Axsun and Santec operating in the 1 μm region, with a 100 kHz repetition rate, which is about three times the range achievable using either source’s built-in k-clock. We have also analyzed the impact the digitization has on the axial range and resolution of the system.

Optical coherence tomography angiography (OCTA) has been widely used for en face visualization of vasculatures but challenged for real 3D topologic imaging due to the ‘tail’ artifacts that appear below large vessel because of multiple scattered light within the vessel. We introduce a normalized field autocorrelation function-based OCTA (𝒈𝟏-OCTA) which minimizes the projection artifacts and is capable of 3D topologic vasculature imaging. 𝒈𝟏(τ) is calculated from repeated OCT acquisitions for each spatial location. The largest decay of 𝒈𝟏(τ) is retrieved to represent the dynamics for each voxel. To account for the small 𝒈𝟏(τ) decay in capillaries where red blood cells (RBCs) are flowing slowly and discontinuously, Intralipid is injected to enhance the OCT signal. With the Intralipid-enhanced signal and shorter decorrelation time processing, we demonstrate that the proposed technique realized 3D OCTA with high signal-to-noise ratio and a negligible ‘tail’ projection. In addition, compared to regular OCTA, the proposed 𝒈𝟏-OCTA doubles the imaging depth. By reducing ‘tail’ artifacts, this technique provides a more accurate rendering of the vascular anatomy for more quantitative characterization of the vascular networks.

In this paper, we extend the master slave (MS) method, so far applied to the modulus of the spectra acquired in spectral domain interferometry, to processing complex spectra. We present the algorithm of complex master slave interferometry (CMSI) method and illustrate the importance of phase processing for signal stability and strength. We demonstrate better stability of the signal driving a direct en-face OCT image by processing both real part and imaginary part of the CMS signal. Then we show that by processing the phase, novel avenues can be opened for the master slave method. A first avenue detailed here is that of dispersion measurements.

Optical coherence tomography (OCT) imaging is a high resolution and non-invasive imaging modality that provides cross-sectional images of a tissue. The time of flight in the signal processing of the OCT system is calculated based on a constant refractive index. Different layers of the complex tissues however have different refractive indices. This issue prompts pixels in the image to be misplaced and that makes it difficult for the physicians to have an accurate diagnosis of abnormalities based on OCT imaging, e.g., in measurement of the thickness of skin layers. In this paper, we propose a novel post-processing method to correct for the refractive index. The proposed method is based on imaging a tissue to which a needle has been penetrated.

A line-field, spectral domain optical coherence tomography (LF-SD-OCT) system was developed for in-vivo, noncontact, cellular resolution imaging of biological tissue. The LF-SD-OCT system utilizes a broadband laser with a spectrum centered at ~790 nm and spectral bandwidth of ~140 nm to achieve 1.8 μm axial and ~5 μm isotropic lateral resolution in biological tissue. A high speed 2D camera was used to achieve frame rate of 2.5k B-scans/s. The system’s SNR was measured to be 92 dB at 100 μm away from the zero-delay line for 2.8 mW optical power incident on the imaged object, with 18 dB roll-off over a scanning range of 1 mm. The LF-SD-OCT system was used to image the cellular structure of cucumber and the cucumber seed where the high spatial resolution was sufficient to resolve cellular nuclei. Then the system was used to image in-vivo human skin (fingertip), where the spiral structures of the sweat glands, as well as a large number of capillaries were observed in the epidermal layer. Images of the healthy human cornea were also acquired from locations near the corneal apex and the periphery and showed the tissue cellular structure and vasculature. Currently, the corneal images were acquired ex-vivo, as we are waiting for ethics clearance to conduct in-vivo corneal imaging studies with the novel LF-SD-OCT system.

Tunable semiconductor laser in red visible spectral range of 670-690 nm is investigated. Swept laser is based on a recently developed traveling wave semiconductor optical amplifier (SOA) of red spectral range as an active element and an acousto-optic tunable filter (AOTF) in an external fiber ring cavity. Tuning band of up to 20 nm, spectral linewidth below 0.04 nm, sweep speed of up to 104 nm/s and CW output power of up to 2.0 mW are obtained. Master-Oscillator Power Amplifier (MOPA) system permitted to increase the output power up to 15 mW with a laser used as a master oscillator and an external SOA - as a power amplifier. We believe the red source may find applications in swept source optical coherence tomography.

MEMS tunable lasers are not inherently phase stable because Brownian motion and drive electronics noise make the starting wavelength of the sweep unstable with respect to the electrical sweep trigger. A typical solution to the problem is to use a fiber Bragg reflector wavelength trigger. That is a sub-optimal solution since environmental changes can move both the Bragg peak and the k-clock phase. We have packaged temperature controlled trigger and clock etalons in a butterfly package to solve this environmental problem. By making the wide FSR trigger etalon from silicon and the narrow FSR clock etalon from fused silica, the relative spectral positions of the trigger and clock can be adjusted through temperature control. The system has applications in background subtraction, phase-sensitive and Doppler sensing, synthetic aperture imaging, and long-term averaging to increase SNR. It can be used for direct hardware clocking of a DAQ board, as well as in a software resampling context.

We investigate the utilization of a high frame rate, 2-D commercial-grade camera in a spectral domain (SD) OCT system driven by a super-luminescent (SLD) light source, using parallel illumination on the sample with a line focus (line-field SD-OCT, LF-SD-OCT). To this goal, several regimes of operation of the camera are evaluated, for different values of the exposure time, ISO and image size, assessing their suitability for depth resolved imaging. A-scans and B-scans of specular and scattering samples are produced, albeit of lesser quality than those we obtained in the past with a relatively expensive, high bit-depth, scientific camera. A comparative study involving several of the camera parameters and their impact on the system’s imaging range and resolution is presented.

Undesirable cross-coupling between polarisation-maintaining (PM) fibers can result in detrimental ghost artefacts within polarisation sensitive optical coherence tomography (PS-OCT) images. Such artefacts combine with coherence noise stripes (originating from Fresnel reflections of optical components), complex-conjugate derived mirror-images and further irregular autocorrelation terms originating from the sample. Together, these artefacts can severely degrade the detected images, making quantitative measurements of the tissue birefringence challenging to perform. In this work, we utilize the recently presented wavelet-FFT filter1 to efficiently suppress these imaging artefacts entirely through post-processing. While the original algorithm was designed to suppress one-dimensional stripe artefacts, we extend this methodology to also facilitate removal of artefacts following a duplicate or inverse (mirror) profile to that of the skin surface. This process does not require any hardware modification of the system and can be applied retroactively to previously acquired OCT images. The performance of this methodology is evaluated by processing artefact-corrupted PS-OCT images of skin consisting of simultaneously detected horizontal and vertical polarized light. The resulting images are used to calculate a phase retardance map within the skin, the profile of which is indicative of localized birefringence. Artefacts in the resulting processed PSOCT images were notably attenuated compared to the unprocessed raw-data, with minimal degradation to the underlying phase retardation information. This should improve the reliability of curve-fitting for measurements of depth-resolved birefringence.

Optical methods have been recently used to perform objective assessment of crystalline lens and corneal opacities. Swept-source optical coherence tomography (SS-OCT) enables measurements of the back-reflected or back-scattered photons from the internal objects. In this work, we present a long-depth range SS-OCT system, with a focus tunable lens, optimized for the visualization of large sections of the posterior segment of the eye, including the vitreous. The system was validated using an eye model.

In this study we combined cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), based on second harmonic generation, and two-photon-excited fluorescence to visualize collagen fibers and tumor cells in the various morphological subtypes of breast cancer. The ability of CP OCT to visualize tissue birefringence and cross-scattering adds new information about the microstructure of such breast cancers, while the MPT provides verification of this microstructure. Mammary glands, both normal and tumorous, were assessed by MPT and CP OCT to establish the relationships between spatial organization features of the cellular component and the intercellular matrix. It was shown, that such multimodal optical imaging has great potential for distinguishing various breast cancer morphological subtypes and could provide useful tools for identifying positive breast cancer margins for surgery.

Lymphatic metastasis is a main pathway of dissemination of malignancies. The diagnosis of metastasis in lymph nodes can help stage cancer or help the surgeons make intraoperative decisions. In addition, lymph nodes are more easily confused with other neck tissues during thyroid surgery. Therefore, identification of lymph nodes is very important. Up to now, the gold standard for identification of metastatic lymph nodes is still histological examination, which can only be performed ex vivo and needs a long time. Optical coherence tomography (OCT) is a non-invasive, high-resolution imaging technology that is capable of detecting microstructures in bio-tissues in real time. In this study, we demonstrated a method to identify metastatic lymph nodes automatically by intraoperative OCT imaging. With a home-made swept source OCT system, we obtained OCT images of different resected neck tissues, including lymph nodes with and without metastasis, thyroid, parathyroid, fat and muscle, from 28 patients undertaking thyroidectomy. The automatic identification algorithm was based on texture analysis and back-propagation artificial neural network (BP-ANN). 66 texture features of OCT images were extracted and 14 were selected and used for automatic identification experiments. The trained BP-ANN has an excellent performance in identifying OCT images of lymph nodes with the sensitivity of 98.9 % and specificity of 98.8 %. The accuracy of lymphatic metastasis diagnosis is 90.1 %.

Spontaneous preterm birth (sPTB) is one of the most serious causes of neonatal death. However, sPTB is unpredictable at present due to simplistic research. That cervix remodels progressively through collagen alterations plays an important role during gestation, but the study of cervical collagen structure has been limited by the lack of suitable observational method. Polarization-sensitive optical coherence tomography (PSOCT) is a functional extension of intensity-based OCT, which can noninvasively offer additional information, i.e., the light’s polarization state. Thus, the collagen properties of birefringence and depolarization can be obtained by a PSOCT in vivo. A PSOCT has been developed from our in-house swept-source (SS) OCT. In the PS-SS-OCT, a circularly polarized light is used to interact tissue and the backscattered light which carries sample’s polarization information is detected by two channels for measuring the horizontal and vertical polarization state respectively. Several human cervix tissues have been investigated by the PS-SS-OCT in vitro. The birefringence and depolarization information of cervical collagen can be obtained by processing the intensity and phase value of the two channels. Besides the birefringence and depolarization information, a conical beam scan strategy has been applied for exploring orientation of the collagen structure of human cervix. In the conical scan, the illumination beam streams into sample at a 45° of incidence angle, and the sample is imaged by acquiring successive B-scan over sample-rotation spans of 0-360°. Since probe of PSOCT can be easily integrated into a catheter or a hand-held probe, PSSS-OCT with a conical beam scan is an excellent candidate to identify cervical structure in clinical practice.

We report a multi-frame superresolution enhanced spectral domain optical coherence tomography (SD-OCT) for fast and high quality in vivo 3D imaging of subsurface fingerprint. After acquiring several sets of low resolution C-scans 3D fingerprint images with lateral sub-spot-spacing shifts, a high lateral resolution and high quality 3D image is reconstructed by multi-frame superresolution processing. In experiment, about 3 times lateral resolution improvement has been achieved from 25 to 7.81 μm with sample arm optics of 0.015 numerical aperture, as well as doubling the image quality. For in vivo 3D SD-OCT imaging, high quality 3D subsurface live fingerprint images have been obtained within a short scan time, showing beautiful and clear distribution of eccrine sweat glands that could be an effective indictor to SD-OCT lateral resolution. Without using any complex segment algorithm, our high quality 3D fingerprint image could be easily separated into three layers: the external fingerprint patterns, the distribution of eccrine sweat glands, and the internal fingerprint pattern. The latter two subsurface layers will benefit high security biometry applications against spoofing attacks.

The index of refraction (n) is an intrinsic parameter of materials and tissues that has recently been proven useful as a biomarker for the diagnosis disease. It can also serve as a source of optical contrast for imaging and provides invaluable information on disease and cell dynamics for studies in various fields such as hematology, oncology, etc. There are many methods to experimentally measure n, e.g. using prisms or interferometers. Optical coherence tomography (OCT) has also been used in the past to measure the index ex vivo. However, the methodologies reported to date are not appropriate for in vivo imaging since they require either a mirror below the sample or an otherwise complicated imaging setup and algorithm. In this summary, we propose a new measurement technique that could be deployed for in vivo estimation of n. This technique uses two OCT images obtained at different incidence angles. The path-lengths observed, in the sample, are different in the two images and directly depend on n. Measuring the path length changes and the incidence angles can provide an estimate of the index. The dual-angle method was validated experimentally using both clear and scattering samples. The resulting measurements of n were within a mean of ~1 % of the expected values. These initial results are promising and provide evidence that this method should be further investigated and validated on human tissues so that, in the future, it could be developed into a clinically useful diagnostic tool.